Studies on the mechanism of mutagenesis in prokaryotic cells have focused on the roles of the RecA and UmuDC-like mutagenesis proteins (which facilitate DNA polymerase bypass of unrepaired DNA lesions). We have overproduced, purified and crystallized the E.coli UmuD' protein. The structure was refined to 2.5 angstroms and analyses revealed that in addition to forming a molecular dimer with itself, the amino terminal of UmuD' can interact with the amino terminal of another "molecular dimer" to form an extended polymer ("filament") structure. While deletion of the N-terminal of UmuD' still allows the protein to form the "molecular dimer", it precludes the formation of the polymer. The N-terminal deletion mutant of UmuD' demonstrates a greatly reduced ability to bind to the RecA nucleoprotein filament and explains why cells expressing the mutant (together with UmuC) are non-mutable. In other studies, construction of chimeric UmuC proteins were generated to investigate the poorly mutable phenotype associated with S.typhimurium. These constructs revealed that alterations in the region located between S.typhimurium UmuC residues 26-59 are most likely the cause of the poorly mutable phenotype. We have also investigated the in vivo stability of the Umu proteins in E. coli. UmuC appears to be inherently unstable, but is partially stabilized in the presence of UmuD. The mutagenically active UmuD' appears to stabilize UmuC further; moreover, UmuC was stabilized still further when RecA protein was constitutively expressed in its activated state. Studies with Xenopus laevis demonstrated that while oocytes can efficiently replicate undamaged single-stranded DNA, they are unable to replicate DNA containing adducts. This replication arrest was alleviated in progesterone-matured oocytes and in oocytes microinjected with mRNAs encoding the prokaryotic UmuD'C or MucA'B mutagenesis proteins, suggesting that the basic mechanisms of mutagenesis are highly conserved between prokaryotic and eukaryotic cells. In a second project, we continued studies on a protein (UV-DDB) which binds to 6/4 photoproducts in UV-damaged human DNA. Purified UV-DDB was not essential in an in vitro nucleotide excision repair system, but it may be required in vivo. UV-DDB moves to a tight association with damaged DNA upon UV treatment of cells; RPA, which also redistributes after UV, is also present in the UV-DDB/DNA complex. The interaction of DDB and RPA enhances the DNA binding of either alone. This interaction, similar to XPA + RPA, places UV-DDB in the initial damage recognition step of DNA repair.